Cadaverine, also known as 1,5-diaminopentane, is an important platform chemical with many industrial applications. Cadaverine serves as the component of polymers such as polyamide or polyurethane, chelating agents, or other additives. In particular, polyamide-5,4 is prepared by polycondensation of cadaverine or succinic acid. Polyamide-5,4 having an annual global market of 3.5 million tons is expected to become a biodiesel alternative to conventional petroleum-based polyamides (Mimitsuka et al., Biosci. Biotechnol. Biochem., 71:2130-2135, 2007; Kind et al., Met. Eng., 12:341-351, 2010). For the production of cadaverine, a renewable, biomass-based carbon source is required.
Cadaverine is a polyamine found in a few microorganisms (Tabor and Tabor, Microbiol Rev., 49:81-99, 1985). In the gram negative bacterium E. coli, cadaverine is biosynthesized from L-lysine by L-lysine decarboxylase (see FIG. 1). There are two forms of L-lysine decarboxylase: one is a constitutive one encoded by the ldcC gene, and the other is an inducible one at low pH, which is encoded by the cadA gene. The level of cadaverine in E. coli is regulated by biosynthesis, degradation, uptake and export (Soksawatmaekhin et al., Mol Microbiol., 51:1401-1412, 2004).
It was reported that cadaverine is not detectable in wild-type E. coli and trace amounts of cadaverine are present in mutants that are defective in the biosynthesis of polyamines (Hafner et al., J. Biol. Chem., 254: 12419-12426, 1979). Although it was reported that a very small amount of cadaverine is present in microorganisms, microorganisms can tolerate a higher concentration of cadaverine. For example, wild-type Corynebacterium glutamicum is able to grow in the presence of about 0.3M cadaverine, although it does not biosynthesize cadaverine (Mimitsuka et al., Biosci. Biotechnol. Biochem., 71:2130-2135, 2007). The high tolerance of microorganisms to cadaverine implies that the microorganisms might potentially be metabolically engineered to overproduce cadaverine to industrially useful levels.
European Patent Publication No. 0726240 A1 discloses a method of producing cadaverine by fermentation using inexpensive industrial waste products or materials having protein and a major constituent. However, there is a problem in that, because the disclosed materials are very complex, many purification steps have to be carried out in order to obtain cadaverine and putrescine. WO 2007/113127 A1 discloses a process of biochemically producing cadaverine using recombinant microorganisms. In this patent publication, in order to increase the conversion of lysine to cadaverine, the activity of lysine decarboxylase is increased by overexpressing lysine decarboxylase encoded by the ldcC gene that is involved in this conversion. In this case, the increase in lysine decarboxylase activity results in an increase in the amount of cadaverine, but the degradation of cadaverine is also induced.
Studies on the degradation and utilization of cadaverine in microorganisms are as follows. Bowman et al. have reported that putrescine/cadaverine aminopropyl transferase which is the product of speE promotes the biosynthesis of aminopropyl cadaverine from cadaverine in E. coli (Bowman et al., J. Biol. Chem., 248:2480-2486, 1973).
Haywood et al. have reported that the yeast Candida boidinii acetylates putrescine to N-acetylputrescine by N-acetyltransferase. It appears that spermidine acetyltransferase which is the E. coli speG gene product has high homology with the N-acetyltransferase of the yeast, and thus possess cadaverine acetyltransferase (Haywood and Large, Eur. J. Biochem., 148:277-283, 1985).
Samsonova et al. have reported that another putrescine degradation pathway involves YgjG putrescine/cadaverine aminotransferase and YdcW dehydrogenase without γ-glutamylation (Samsonova et al., BMC Microbiol., 3:2, 2003; Samsonova et al., FEBS Lett., 579:4107-4112, 2005).
Kurihara et al. has called the putrescine degradation pathway as “Puu catabolic pathway” based on the finding that the putrescine degradation pathway is closely associated with γ-glutamylated metabolites of E. coli. This pathway appears to be also involved in cadaverine degradation. For example, glutamate-putrescine/glutamate-cadaverine ligase encoded by the puuA gene that is involved in this pathway can covert cadaverine into γ-glutamyl-L-cadaverine. Additionally, a putrescine importer which is the product of the puuP gene is associated with the catabolic pathway and major putrescine importers (Kurihara et al., J. Biol. Chem., 280: 4602-4608, 2005). It can be thought that such putrescine importers introduce cadaverine, because cadaverine is structurally similar to putrescine.
Accordingly, the present inventors have prepared a mutant microorganism wherein at least one gene selected from the group consisting of a speE gene encoding putrescine/cadaverine aminopropyl transferase, a speG gene encoding spermidine N-acetyltransferase, a ygjG gene encoding putrescine/cadaverine aminotransferase, a puuP gene encoding putrescine importer, and a puuA gene encoding glutamate-putrescine/glutamate-cadaverine ligase, which are involved in the cadaverine degradation or utilization pathway of cadaverine-producing microorganisms, is inactivated or deleted, and have found that, when the mutant microorganism is cultured, it can produce cadaverine in high yield, thereby completing the present invention.